JPH06128296A - Method of preparing human antithrombin -iii - Google Patents

Abstract

PURPOSE: To provide an easy and effective method for purifying human antithrombin-III existing in an alcohol-containing solution.

CONSTITUTION: Human antithrombin-III (AT hereafter) is purified by an affinity- chromatography method characterized by bringing an alcohol-comprising solution containing human AT-III into contact with solid phase combining heparin at a low temperature of 3°C to -10°C to combine the AT-III to heparin. This process makes it possible to purify human AT-III easily and efficiently.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for preparing human antithrombin-III.

[0002]

2. Description of the Related Art Antithrombin-III (hereinafter referred to as AT-
(Referred to as III) is α ₂ -globulin known to suppress blood coagulation. AT-III acts essentially as an irreversible suppressor. AT-III is believed to suppress serine proteases in plasma during activation of either the coagulation system or the fibrinolytic system. AT
-III has an important effect of suppressing factor Xa and thrombin. A family with congenital AT-III deficiency
The incidence of thrombosis is high. Attempts have been made to administer AT-III to patients with thrombotic disorders.

Several attempts have been reported to purify AT-III from human plasma and plasma pastes using conventional techniques. However, these methods require a lot of time and have a low yield. The purification method was substantially improved by incorporating an affinity chromatography step using purified heparin as the solid phase bound ligand (ligand).

Dams and Wallace (Biochem. Biophys. Res.
Comm., 61 (4); 1147, 1974) purified canine AT-III by a method incorporating heparin-sepharose chromatography. In this method, plasma from which fibrin has been removed by heat treatment is passed through a small column containing heparin-sepharose at 8 ° C. However, when the distribution of specific activity was measured over the elution profile, the final product still contained foreign material. Miller-Anderson et al. (Thromb. Res., 5: 439, 1
974) used human heparin-sepharose
Discloses to purify III. In this method, citrate-added human plasma frozen immediately after removing blood cells by centrifugation is added to heparin-sepharose in batch at 5 ° C. Then, this gel-like suspension is injected into the column and allowed to settle, and the adsorbed substance is eluted with a salt gradient. The yield of this whole process (including ion exchange and gel filtration chromatography) was 34%. However, this method makes large-scale production of AT-III difficult because of the large number of chromatographic separations. Thaler and Sumer (Br. J. H
aemat., 31: 233, 1975) show a method for isolating human and bovine AT-III involving heparin-agarose chromatography and polyethylene glycol precipitation. This method is all performed at 4 ° C. Either chromatography or precipitation can be the first step of the purification process. Wickelhauser, etc. (Vox Sanq.,
36: 281, 1979) show a large-scale method for preparing AT-III from plasma or Cohn fraction IV-1. This method involves first performing polyethylene glycol precipitation, then batch adsorbing to heparin-sepharose, desalting by ultrafiltration, and then pasteurizing the final product. Recovery by activity was 32% from plasma and 16% from Cohn Fraction IV. All purification steps are performed at 5 ° C.

[0005]

An object of the present invention is to provide a simple and effective method for purifying human AT-III present in an ethanol-containing solution. Another object of the present invention is to use AT-II which uses immobilized heparin as a ligand.
The I purification method is applied to an ethanol solution containing AT-III. Still another object of the present invention is to obtain purified AT-III from supernatant II + III obtained by the Cohn fractionation method.

[0006]

The above and other objects provide a method of contacting an alcohol-containing solution containing AT-III such as ethanol-containing human plasma supernatant with a heparin affinity gel matrix at a temperature of 4 ° C. or lower. The above-mentioned temperature is preferably 0 ° C. or lower. That is, the present invention relates to the following (1) and (2). (1) Human antithrombin-III comprising the following steps
Preparation method of. (a) An alcohol-containing solution of human plasma containing human antithrombin-III is obtained, and (b) the alcohol-containing solution is contacted with solid phase-bound heparin at 3 ° C to -10 ° C to form a mixture, and antithrombin- III is bound to the heparin, (c) the solid phase-bound heparin is separated from the solution, (d) the human antithrombin-III is eluted from the solid phase-bound heparin to obtain an antithrombin-III solution. . (2) Human antithrombin-II comprising the following steps
A method for purifying an alcohol-containing solution containing I and other proteins. (a) contacting the alcohol-containing solution with solid phase-bound heparin at 3 ° C to -10 ° C to form a mixture, allowing antithrombin-III to bind to the heparin, and (b) adding the solid phase-bound heparin to the solution. And (c) the human antithrombin-III is eluted from the solid phase-bound heparin to obtain a purified antithrombin-III solution.

The human plasma fraction containing alcohol is the preferred starting material for use in the present invention. For example, supernatant II + III by the Cohn fractionation method, or supernatant I, a residual fraction after collecting blood coagulation factor VIII from citrate-containing plasma, and the like are exemplified. Of these, supernatant II + III obtained by the Cohn fractionation method is preferable. Supernatant II + III from the Cohn fractionation method contains approximately 20% ethanol.

The heparin is a monomeric heparin bound to an insoluble solid support consisting of, for example, polysaccharides, silica gel or fibers. Examples of such solid supports include agar, agarose, crosslinked agarose, cellulose,
Examples include silica, nylon and others known in the art.

In the method for purifying human AT-III of the present invention, the alcohol-containing plasma fraction is applied to a heparin gel matrix at about 3 ° C to about -10 ° C, preferably 0 ° C or lower, more preferably 0 to -6 ° C. And more preferably at about -6 ° C. When the above-mentioned purification is carried out in a batch system, the ligand-bound AT-III is recovered by a conventional method such as centrifugation.
It can be separated from the mixture of II + III and gel matrix.

AT-III is washed with 0.1 to 1 M NaCl or the like, and particularly preferably after the washing operation is repeated by gradually increasing the salt concentration, and then the AT-III is added to a high salt concentration buffer such as 2 M NaCl. It is eluted from the heparin-gel matrix by conventional methods such as contacting.

The purified AT-III solution contains, for example, pH.
About 7-8 in about 0.2M to about 0.6M citrate buffer for about 10 hours, heat treatment at about 60 ° C, or detergent at about 20 ° C to about 40 ° C for about 30 minutes to about 10 hours. The virus present may be inactivated by contacting it and then performing a detergent treatment to remove the detergent by a conventional method such as chromatography. For example, Horowitz et al. (Transfusion, 25 (6): 51
6, 1985) is tri (n-butyl) phosphate (TN
It has been disclosed to inactivate the virus using a detergent such as BP) or Tween 80.

[0012] In one embodiment, the supernatant II + III obtained by the Cohn fractionation method is treated with heparin-sepharose (Pharmacia).
And heparin- Actigel Superflow (manufactured by Sterogene) are mixed by a batch adsorption method, and the suspension is kept at a low temperature of 3 ° C. or lower, preferably 0 to −6 ° C. Prior to contacting the solid phase heparin with the plasma fraction, the heparin-sepharose is washed with an alcohol-containing solution, for example an alcohol and a concentration of 20% ethanol solution suitable for use in the present invention. In addition, a 25% ethanol solution is suitable as the washing solution. The heparin-sepharose is then separated from the liquid phase. In another embodiment, the column is loaded with heparin-solid phase matrix and the plasma alcohol fraction is passed through the column. Again, solid phase heparin may be added, for example, 20-2 before and after column packing.
Wash with an alcohol containing solution such as a 5% ethanol solution. Other important proteins such as albumin, haptoglobin, α-1 protease inhibitor, and IgA, which are denatured in the presence of alcohol at 4 ° C or higher when supernatant II + III obtained by the Cohn fractionation method is treated at a low temperature of 4 ° C or lower. Is not affected. The alcohol is also useful in preventing the solid phase from freezing under the low temperatures applied in the separation process. Further, by subjecting it to hydrophobic chromatographic treatment, anion exchanger treatment and the like, contaminants such as other plasma proteins, pyrogens and heat denatured proteins are removed to obtain highly pure AT-
III can be purified and recovered (JP-A-1-275).
No. 600, JP-A-2-4717).

[0013]

The present invention will be described in detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 Disclosure of corn and the like (J. Am. Chem. Soc., 72, 465 (1950))
According to the procedure, supernatant II + III was obtained from human plasma by corn fractionation at -4 ° C to -6 ° C. Heparin-Actigel Superflow
(Manufactured by Sterogene) was washed with a 20% ethanol solution at −4 ° C. to −6 ° C. and packed in a 100 ml column at −6 ° C. Then, the supernatant II + III 1500 by the Cohn fractionation method
ml was passed through the column. After the above operation, the column was washed with 50 ml of 20% ethanol. This washed fraction was processed by the method to obtain corn albumin. The column was washed with 0.3 M NaCl solution to remove non-specifically bound proteins. AT-III was eluted with a 2M NaCl solution. The above AT-III was subjected to ultrafiltration (Filtron, O
mega, 10K). Virus inactivation treatment includes 1 v / v% Tween 80 and 0.3 v / v% TN
BP was added to the above concentrated AT-III solution and kept at 30 ° C. for 6 hours. The detergent was removed by ion exchange chromatography (DEAE-Sephadex or heparin affinity chromatography). Above AT
The III solution (50 u / ml) was lyophilized under sterile conditions.

A from supernatant II + III by Cohn fractionation
The T-III yield is 60%, and albumin, globulin, transferrin and other contaminating proteins have a cellulose acetate membrane (Jowkin, M. et al., PNAS, USA 7
6, 4350 (1978)) and polyacrylamide (Laemmli,
It was not detected by electrophoresis in UK et al., Nature, 227, 680 (1970).

Example 2 Heparin washed with 20% ethanol at -6 ° C
To 200 ml of Sepharose (Pharma-cia), 2 liters of the supernatant II + III by the Cohn fractionation method was mixed at -4 ° C,
The mixture was stirred at -4 ° C for 60 minutes. The heparin-sepharose was collected by filtration and 100 ml of 20% ethanol at -4 ° C followed by 0.3M NaCl at -4 ° C.
Washed with 500 ml. AT-III was eluted in the same manner as in Example 1. As a virus inactivating treatment, AT-II obtained by concentrating 0.5 M sodium citrate by ultrafiltration was used.
I solution was added. The solution was adjusted to pH 7.5 and then heated to 60 ° C. for 10 hours. After the above heat treatment, sodium citrate was removed by ultrafiltration. AT-
The III solution (50 u / ml) was lyophilized under aseptic conditions.

A from supernatant II + III by Cohn fractionation
The T-III yield was 70% after heparin treatment and 55% after heat treatment. Albumin, globulin, transferrin and other contaminant proteins were not detected by electrophoresis.

Example 3 Washing with a heparin-sepharose treatment of 0.7M
Same as Example 2 except that NaCl was used. 60 ° C, 10
After heat treatment for hours, sodium chloride (final concentration 3M)
And sodium citrate (final concentration 20 mM) were added to adjust the pH to 7.5. Meanwhile, 20 mM sodium citrate buffer containing 3 M sodium chloride (pH 7.5)
The anti-thrombin-III-containing aqueous solution was brought into contact with the butyl-type polyvinyl carrier (Butyl-Toyopearl 650, manufactured by Toyo Soda Co., Ltd.) equilibrated with, and developed with the above-mentioned buffer solution, and the unadsorbed fraction was recovered. Subsequently, the solution was concentrated overnight while being dialyzed against a 0.9% sodium chloride solution to obtain a 1 (w / v)% aqueous solution of antithrombin-III, which was clarified by filtration or centrifugation as necessary. It was a liquid. 2% (w / v) mannitol and 0.2% sodium citrate were added to a 1 (w / v)% aqueous solution of antithrombin-III.
(W / v)% was added, diluted with a small amount of cold distilled water so that sodium chloride became 0.5%, adjusted to pH 7.6 with 1N sodium hydroxide, and then sterilized with a sterilized Millipore filter. Then, 500 units were dispensed and freeze-dried to obtain a dry preparation.

It will be apparent to those skilled in the art that the present invention can be modified without departing from its spirit. Modifications that do not depart from the spirit and scope of the appended claims are considered equivalent thereto.

Front Page Continuation (72) Inventor Swarai Kawool United States, 91763 Vermon Avenue, Moncler, CA 10395 (72) Inventor Prabil Batchaea U.S.A., 91789 Mulfield Lane, California 300 (72) Inventor Large Mizusho Tadashi 2-25-1 Otani Otani, Hirakata-shi, Osaka Prefecture Midori Cross Central Research Institute Co., Ltd. (72) Inventor Yoshio Itagaki 3-5-44, Tsushima Nakadori, Miyakojima-ku, Osaka City Midori Cross Miyakojima Factory Within

Claims (23)

[Claims] 1. A method for preparing human antithrombin-III comprising the following steps. (a) Human antithrombin-III
To obtain an alcohol-containing solution of human plasma containing (b) 3 ° C.
The above alcohol-containing solution is contacted with solid phase bound heparin at -10 ° C to form a mixture, antithrombin-III
To the heparin, (c) the solid phase-bound heparin is separated from the solution, and (d) the human antithrombin-III is eluted from the solid phase-bound heparin to obtain an antithrombin-III solution. 2. The method according to claim 1, wherein the antithrombin-III solution is subjected to virus inactivation treatment. 3. The method according to claim 2, wherein the virus inactivating treatment is heat treatment. 4. The method according to claim 2, wherein the virus inactivation treatment is detergent treatment. 5. The method according to claim 1, wherein the alcohol-containing solution of human plasma is supernatant II + III obtained by the Cohn fractionation method. 6. The method according to claim 1, wherein the contacting step (b) is performed at 0 ° C. or lower. 7. The method according to claim 1, wherein the contacting step (b) is carried out at 0 to −6 ° C. 8. The method of claim 1 wherein said contacting step (b) is performed at about -6 ° C. 9. The method of claim 1, wherein the alcohol-containing solution of human plasma contains ethanol. 10. A method for purifying an alcohol-containing solution containing human antithrombin-III and other proteins, which comprises the following steps. (a) contacting the alcohol-containing solution with solid phase-bound heparin at 3 ° C to -10 ° C to form a mixture, allowing antithrombin-III to bind to the heparin, and (b) adding the solid phase-bound heparin to the solution. And (c) the human antithrombin-III is eluted from the solid-phase bound heparin to obtain purified antithrombin-III.
Get a solution. 11. The method of claim 10, wherein the alcohol is ethanol. 12. The method according to claim 10, wherein the alcohol-containing solution is supernatant II + III obtained by the Cohn fractionation method. 13. The method according to claim 12, wherein the contacting step (a) is performed at 0 ° C. or lower. 14. The method according to claim 12, wherein the contacting step (a) is carried out at 0 to −6 ° C. 15. The method of claim 12 wherein said contacting step (a) is performed at about -6 ° C. 16. The method according to claim 12, wherein the solid phase-bound heparin is washed with an alcohol-containing solution before the contacting step (a). 17. The method according to claim 16, wherein the washing is performed using a 20 to 25% ethanol aqueous solution. 18. The method according to claim 12, wherein the antithrombin-III solution is subjected to virus inactivation treatment. 19. The method according to claim 18, wherein the virus inactivating treatment comprises heat treatment or detergent treatment. 20. A purified human antithrombin-III solution obtained by the method according to claim 12. 21. A solution of human antithrombin-III obtained by the method according to claim 5. 22. In step (d), after washing the solid phase-bound heparin with a 0.1 to 1 M aqueous sodium chloride solution,
The method according to claim 1, wherein human antithrombin-III is eluted from the solid-phase bound heparin. 23. In step (c), after washing the solid phase-bound heparin with a 0.1 to 1 M aqueous sodium chloride solution,
The method according to claim 10, wherein human antithrombin-III is eluted from the solid-phase bound heparin.